A large number bacterial pathogens cause disease via the translocation of proteins that misregulate normal host cell function. Many Gram-negative bacteria use specialized protein translocation systems to accomplish this goal. Chief among these are the Type III Secretion Systems, which are critical for causing diseases as common as enteropathogenic diarrhea and as virulent as bubonic plague. These protein translocation systems have a number of conserved components found in a broad swath of pathogens, so they are potential sites for therapeutic intervention. Unfortunately, our understanding of the functional details of specialized protein translocation systems is extremely limited, and very little is known about the events that occur after attachment of the bacterium to the host cell and prior to the encounter of the translocated protein with its target. This proposal is intended to address this gap, and set the stage for a long-term study that determines how the host cell facilitates the deposition and function of protein substrates that move from the bacterium, via the specialized secretion system, into the host cell. As there are very few leads regarding how to approach this long-term goal, this application proposes to develop a strategy that has the potential to identify every host cell protein that modulates the function of a translocated substrate. For this purpose, the translocation of a single bacterial protein substrate will be analyzed in detail, with the eventual goal of identifying host proteins that interface with a large number of proteins deposited by a broad range of pathogens. Two assay strategies are proposed that should allow high throughput identification of host proteins that support the activity of the Yersinia pseudotuberculosis YopE protein, a translocated substrate of all Type III Secretion Systems encoded by enteropathogenic Yersinia and the causative agent of bubonic plague. Each takes advantage of the ability to identify host cells that are unable to support YopE function, which can be detected by an increase in fluorescence resonance energy transfer readout. In each case, host cells subjected to a specific interfering RNA (RNAi) treatment that reduces YopE activity will be identified based on this readout, and identified RNAi molecules will be analyzed further in secondary assays. These secondary assays will determine whether the host protein is required for movement of YopE out of the bacterium into the host cell, movement of YopE to find its host cell target, or recognition of target. Most importantly, the proposed work has the potential to identify a host cell system that escorts Type III Secretion System substrates to their target. Disruption of such a relationship is potentially an important site for future therapeutics directed against bacterial pathogens. PUBLIC HEALTH RELEVANCE: Specialized protein translocation systems encoded by pathogenic bacteria are critical for establishing colonization and causing disease within human hosts by facilitating the movement of bacterial proteins that are toxic for host cells. Evidence exists that the host cell is somehow collaborating with the pathogen to allow itself to be intoxicated, but unfortunately very little is known regarding how the host becomes an unwitting participant in the process. By taking advantage of assays recently developed for identifying cells misregulated by the bacterial protein, schemes are proposed to uncover proteins that collaborate with the pathogen. The data obtained are intended to work toward the long-term goal of finding host proteins that escort a broad swath of pathogen proteins, providing potential sites for chemotherapeutic intervention.